NCB-20, a cloned hybrid cell line of mouse neuroblastoma and fetal Chinese hamster brain cell, has been used as a model system for the study of receptor-receptor interactions in the same cell. NCB-20 cells have 5-HT receptors which are linked to adenylate cyclase. The plasma membranes of these cells also contain high affinity binding sites for 3H-ketanserin and 3H-mianserin. In addition, these cells are equipped with 5-HT presynaptic components. These include a 5-HT uptake system and a very high Bmax of 3H-imipramine binding sites (50-times the Bmax in the CNS). 5-HT can release acetylcholine from NCB-20 cells. When phosphoinositides in the cells were prelabeled by preincubation with 3H-inositol, the addition of carbachol enhances the accumulation of 3H-inositol monophosphate (in the presence of Li+) by about 400%. Significant increase of 3H-inousitol bisphosphate formation can also be detected. This carbachol-induced activation of phospholipase C is time dependent, dose-related (EC50 10 minus 5M) and blocked by atropine. These data provides the first evidence for the presence of functional muscarinic acetylcholine receptors in NCB-20 cells and suggests that acetylcholine released by 5-HT may autoregulate the hydrolysis of phosphoinositides. The information obtained in this model system may gain insight of the molecular mechanism of receptor-receptor communications in the same neuron.